This invention relates generally to a novel assay method. More particularly, it relates to an assay method for the detection of total bilirubin in serum and other body fluids.
Bilirubin is present in biological liquids in different molecular forms, generally divided however into two types, i.e., conjugated and non-conjugated. Assay methods have been devised for detection of conjugated bilirubin, also referred to as direct bilirubin as well as for total bilirubin which includes both the conjugated and non-conjugated forms. Detection of the latter comprises the subject matter of this invention.
Total bilirubin is usually measured in blood serum by means of a diazo reaction in which bilirubin which has been liberated from the protein to which it is bound is reacted with a diazonium salt which leads to the formation of a dypyrrole pigment.
The prior art methods of assaying total bilirubin have numerous shortcomings. For instance, the diazonium salt reagent used in the diazotization reaction is unstable and it is particularly difficult to store either in reconstituted or lyophilized condition. Another common and frequent problem deals with the turbidity problem one encounters when adding the biological liquid to previously used reagents. Such turbidity interferes with the measurement of total bilirubin giving questionable results.
It is the objective of the herein disclosed invention to provide a bi-reagent total bilirubin procedure which can be used in wet or lyophilized chemistry formulations involving a singular reaction effected concurrently or sequentially.
It is a further objective to provide a procedure for assaying total bilirubin in a sample under conditions which overcome the aforedescribed stability and turbidity problems associated with prior art techniques.